SACCHAROMYCES-CEREVISIAE GCS1 IS AN ADP-RIBOSYLATION FACTOR GTPASE-ACTIVATING PROTEIN

Movement of material between intracellular compartments takes place th rough the production of transport vesicles derived from donor membrane s, Vesicle budding that results from the interaction of cytoplasmic co at proteins (coatomer and clathrin) with intracellular organelles requ ires a type of GTP-binding protein termed ADP-ribosylation factor (ARF ). The GTPase cycle of ARF proteins that allows the uncoating and fusi on of a transport vesicle with a target membrane is mediated by ARF-de pendent GTPase-activating proteins (GAPs). A previously identified yea st protein, Gcs1, exhibits structural similarity to a mammalian protei n with ARF-GAP activity in vitro. We show herein that the Gcs1 protein also has ARF-GAP activity in vitro using two yeast Arf proteins as su bstrates. Furthermore, Gcs1 function is needed for the efficient secre tion of invertase, as expected for a component of vesicle transport. T he in vivo role of Gcs1 as an ARF GAP is substantiated by genetic inte ractions between mutations in the ARF1/ARF2 redundant pair of yeast AR F genes and a gcs1-null mutation; cells lacking both Gcs1 and Arf1 pro teins are markedly impaired for growth compared with cells missing eit her protein. Moreover, cells with decreased levels of Arf1 or Arf2 pro tein, and thus with decreased levels of GTP-Arf, are markedly inhibite d for growth by increased GCS1 gene dosage, presumably because increas ed levels of Gcs1 GAP activity further decrease GTP-Arf levels. Thus b y both in vitro and in vivo criteria, Gcs1 is a yeast ARF GAP.